Our long-term objective is to understand better the regulation of the initial steps of spermatogenesis, namely spermatogonial proliferation and differentiation In the current granting period, we demonstrated that proliferation of type A spermatogonia is stimulated by stem cell factor (SCF) Other studies demonstrated that Sertoli cell-spermatogonial co-cultures allow for spermatogonial differentiation, i.e., the isolated type A (presumably stem cells) divided a number of times to yield rows of interconnected differentiated type A. We also established that the PI-3 kinase/AKT/p70 S6 kinase signaling pathway is involved in SCF-induced proliferation of type A spermatogonia. In addition, we demonstrated that differentiation of type A into sperm is associated with changes in telomere length and telomerase activity. In this competing renewal application, in aim 1, we will further examine the biology of the type A spermatogonia. We hypothesize that in addition to SCF, other ligands such as leukemia inhibitory factor (LIF) and glial cell line-derived neurotrophic factor (GDNF) may be important for spermatogonial development. Our preliminary results indicate that receptors (GFRalpha1 and LIFr) for these two ligands are expressed on the surface of type A spermatogonia. We will use whole mount immunocytochemistry to examine whether all type A spermatogonia possess the GFRalpha1, c-kit, and LIF receptors. Using the immunomagnetic bead procedure, we will separate a GFRalpha1 positive group of cells as well as a c-kit and LIFr positive group of cells and stimulate each group with the respective ligands. To determine whether these subsets of type A spermatogonia retain their ability to differentiate, we will transplant them into sterile recipient mice and follow spermatogenesis in vivo. In aim 2, we will continue to investigate the molecular signals responsible for spermatogonial proliferation. We have generated exciting preliminary data to suggest that SCF and LIF may initiate distinct intracellular signaling pathways. Through these signaling pathways, specific candidate genes are induced by ligands leading to spermatogonial proliferation. In the third aim, we will examine gene expression in isolated type A spermatogonia and in seminiferous tubules in culture using serial analysis of gene expression (SAGE) and DNA array procedures in response to ligands. The experiments outlined in this proposal should provide new data on the basic biology of the spermatogonia. Furthermore, these studies will help in developing techniques of in-vitro spermatogenesis and contribute to the use of healthy germ cells in assisted reproduction.